WO1998017623A2 - Inhibiteurs du transport de la polyamine - Google Patents

Inhibiteurs du transport de la polyamine Download PDF

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Publication number
WO1998017623A2
WO1998017623A2 PCT/IB1997/001651 IB9701651W WO9817623A2 WO 1998017623 A2 WO1998017623 A2 WO 1998017623A2 IB 9701651 W IB9701651 W IB 9701651W WO 9817623 A2 WO9817623 A2 WO 9817623A2
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WIPO (PCT)
Prior art keywords
polyamine
synthetic derivative
spermine
methyl
desc
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PCT/IB1997/001651
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English (en)
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WO1998017623A3 (fr
Inventor
Richard Poulin
Marie Audette
René CHAREST-GAUDREALT
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Universite Laval
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Priority to AU57752/98A priority Critical patent/AU5775298A/en
Priority to EP97953991A priority patent/EP0876327A2/fr
Publication of WO1998017623A2 publication Critical patent/WO1998017623A2/fr
Publication of WO1998017623A3 publication Critical patent/WO1998017623A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/41—Y being a hydrogen or an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups

Definitions

  • the present invention relates to a novel class of competitive inhibitors of natural polyamine transport in mammalian cells.
  • the present invention is more particularly directed to low molecular weight, high-affinity, specific, impermeant, pure antagonists of polyamine transport of a structure different to that of endogenous polyamines.
  • the novel inhibitors of the present invention exhibit an effect on cultured tumor cells essentially cytostatic, with minor non-specific effects.
  • the present invention is also directed to the use of such novel inhibitors of polyamine transport to evaluate the antitumor efficacy of polyamine depletion strategies with minimal systemic cytotoxic effects or to control and treat disorders involving unrestrained cell proliferation and/or cell differentiation wherein polyamine transport is required.
  • Natural polyamines such as putrescine (1,4-butane-diamine), spermidine (N-3[- aminopropyl]-l,4-diaminobutane) and spermine (N,N'-bis-[3-aminopropyl]-l,4-butane- diamine) play essential roles in the control of macromolecular synthesis and growth processes in eukaryotic cells.
  • Cells maintain appropriate polyamine concentrations principally by de novo synthesis from amino acids wherein ornithine decarboxylase catalyzes conversion of ornithine to putrescine, which is then converted to spermidine and spermine.
  • Most tissues also possess a specific plasma membrane transport system allowing for utilization of plasma sources of polyamines.
  • Inhibitors of polyamine biosynthesis such as a-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, cause an extensive depletion of polyamines followed by growth arrest in virtually all known mammalian cell types in vitro. Since tissues such as tumor cells and other transformed or rapidly proliferating cells exhibit a high demand for polyamines, these properties have encouraged an extensive assessment of such compounds for the treatment of proliferative diseases, including several types of tumors, in experimental models and in clinical trials. Unfortunately, the antitumor efficacy of such inhibitors in vivo has been disappointing. The failure of DFMO to halt tumor growth in animal models has been clearly correlated with the elevated polyamine transport activity found in transformed cells.
  • DFMO a-difluoromethylornithine
  • polymers of Aziz et al. as well as their charge would cause their adsorption to the cellular surface, which bears negative charges due to the presence of glycoproteins, e.g. sialic acid.
  • Poly-L-Lysine a commercially used compound analogous to high molecular weight polymers of polyamines by its positive charges, is known to promote a strong electrostatic interaction between the cell and its substrate, as in the induction of positive charges of gamma irradiation of synthetic polymers used to produce dishes for tissue culture.
  • the polyamine transport inhibitors of Aziz et al. present the additional drawback of being highly cytotoxic. It is noteworthy that their spermine polymer is effective in decreasing contents of polyamines in cells even when not used in combination with DFMO and at concentrations much higher than those required to block polyamine uptake, which indicates inherent high toxicity of the compound toward the cell by a mechanism independent of polyamine transport per se. The cytotoxicity of the spermidine polymer of Aziz et al. is most probably explained by a non-specific effect on cellular physiology such as the cellular membrane.
  • Cysteamine and aliphatic monoamines of similar chain length such a n-butylamine and n-pentylamine have a low but significant ability to antagonize putrescine uptake (Gordonsmith, R.H., Brooke-Taylor, S., Smith, L.L. and Cohen, G.M. 1983. Biochem.
  • Plasma polyamines are partly derived from various dietary sources (7, 12, 18, 58-60, 62, 70) and from the activity of the gastrointestinalmicroflora, wihch produces and excretes very high amounts of putrescine and cadaverine (1, 17, 45, 50, 62, 70), which can enter the generalcirculation thorugh the enterohepatic pathway (6, 45). Other systemic contributions can also be attributed to polyamine excretion by peripheraltissues,including dying tumor cells (32, 35, 41, 42, 63, 64, 67, 79, 80).
  • the enhanced uptake of polyamines by tumor cells results both from the increased polyamine transport activity that accompanies the malignant phenotype (11, 43, 51, 68, 69), and from the effect of DFMO itself, which causes a compensatory upregulation of polyamine uptake across the plasma membrane (9, 10, 14-16, 22, 25, 29, 31, 38, 39, 43,
  • polyamine transport inhibitors having a low molecular weight, less susceptible to immunogenicity and to nonspecific interactions with the cellular membrane. These inhibitors have high affinity, are specific, impermeant, pure antagonists of polyamine transport in mammalian cells while exhibiting minimal cytotoxic effects.
  • the synthetic derivative compromises a dimer wherein monomers of said dimer are linked together by a spacer side chain anchored to the amido group of each monomer.
  • natural polyamines such as putrescine, spermine and spermidine can be used as the original polyamine, other non natural polyamines can be used as a starting material for the making of synthetic derivatives as thought by the present invention. Accordingly, a synthetic derivative comprising the following general formula
  • R ⁇ and R independently represent a hydrogen atom or an alkyl group having 1 to 2 carbon atoms
  • R 2 , R 2 , or R 3 and R 3 independently represent a hydrogen atom or a methyl group
  • w and z independently represent an integrer of 2 or 3
  • x represents an integrer from 0 to n
  • n represents an integrer from 3 to 6
  • S represents a hydrogen atom or a molecule which cannot be captured by said natural polyamine transporter.
  • the side chain S may be labeled and be used as a marker for a polyamine transporter.
  • the side chain S can be varied to increase the affinity of the derivative for the transporter.
  • the side chain S may also become a spacer molecule useful in the formation of a dimer.
  • This spacer side chain comprises a linear hydrocarbon- containing backbone of 3 to 8 atoms.
  • the backbone may comprise sulfur, oxygen, or nitrogen atoms.
  • the original polyamine is spermine.
  • Three derivatives have been obtained therefrom: N-(2-mercaptoethyl)spermine-5-carboxamide (MESC), the disulfide from thereof, namely 2,2 1 -dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), and N-[2,2'-Dithio(Ethyl, 1 '-Aminoethy lspermine-S-carboxamide (DEASC).
  • This inhibition results in the control of the treatment of disorders involving unrestrained cell proliferation and/or differentiation where control of polyamine transport is required, when used in combination with an inhibitor of polyamine synthesis such as DFMO.
  • the above sequence of steps results in the diagnosis of a disorder involving unrestrained cell proliferation and/or differentiation where control of polyamine transport is required.
  • this composition also comprises an inhibitor of polyamine synthesis, such as DFMO.
  • an inhibitor of polyamine synthesis such as DFMO.
  • the applicants have unexpectedly discovered that the presence of a lateral amido group immediately linked to a carbon atom of the polyamine backbone of a synthetic derivative of an original polyamine confers impermeant properties to the so derived synthetic polyamine against the mammalian cell. It follows that the synthetic polyamine derivatives of the present invention, by exhibiting high affinity for diamine and polyamine transport systems, block the transport of natural polyamines by competing therewith, while in the same time acting as poor substrate for intracellular uptake. The affinity of the polyamine derivative for the transporter system is further enhanced by increasing the length of a side chain anchored to the amido group of the derivative.
  • the best affinity is achieved by dimerizing the polyamine derivative with the aid of a spacer molecule anchored at both ends to the amido group of each monomer.
  • the flexibility of the chemical structure of the inhibitors of the present invention permits better optimization of the activity and affinity than a simple polymeric structure such as (TS) n .
  • modifications to the polyamine backbone as taught by the present invention such a methylation of C 1 and C 12, lowers the possibility of oxidation of the primary amides by the serum amine oxidase, which is present in mammalian sera.
  • FIG 2 graphically illustrates the inhibition of [ 14 C]spermine transport by MESC, DESC and DEASC in human ZR-75-1 breast cancer cells.
  • the rate of spermine uptake was measured in ZR-75-1 cells grown as monolayers in 24-well culture plates in the presence of the indicated concentrations of DESC (O), MESC (•), and DEASC (D), using l ⁇ M [ 14 C]spermine as substrate.
  • Data are the mean ⁇ SD of triplicate determinations;
  • FIG 3 graphically illustrates the inhibition of [ 3 H] spermidine uptake by spermine and DESC in ZR-75-1 cells.
  • the rate of spermidine uptake was measured in ZR-75-1 cells grown as monolayers in 24-well culture plates in the presence of the indicated concentrations of spermine (O) and DESC (•) using 3 ⁇ M [ 3 H]putrescine (A) or 1 ⁇ M [ H]spermidine (B) as substrate.
  • Data are the mean ⁇ SD of triplicate determinations from a representative experiment
  • FIG 4 illustrates graphically the Lineweaver-Burke analysis of putrescine transport inhibition by DESC and DEASC in ZR-75-1 cells.
  • FIG 5 illustrates graphically the structure of MESC thioether derivatives and their Kj values with respect to spermidine uptake in CHO-Kl cells.
  • FIG 6 graphically represents the effect of DESC and MESC on the intracellular accumulation of [ 3 H] spermidine in ZR-75-1 cells, wherein at time 0 (A), 5 ⁇ M
  • [ 3 H]spermidine was added to ZR-75-1 cell cultures grown in 24-well plates (lml/well) in the presence of 200 ⁇ M MESC (•), 50 ⁇ M DESC (D) or 200 ⁇ M DESC ( ⁇ ), and accumulation of radiolabeled spermidine determined after the indicated interval.
  • Control cells (O) received vehicle only.
  • B same as in A, except that 200 ⁇ M CHX was added at time 0 in the presence of 0 (•), 50 (D) or 200 ⁇ M DESC ( ⁇ ).
  • Data are the mean ⁇ SD of triplicate determinations;
  • FIG 7 illustrates the effect of spermine, MESC, DESC and DEASC on ZR-75-1 cell proliferations.
  • Cells were incubated for 11 days in MEZR medium with the indicated concentration of spermine, DESC, MeSC, or DEASC in the presence (shaded bars) or absence (plain bars) of 1 mM of aminoguanidine, and DNA content per culture was then determined.
  • Data represent the mean ⁇ SD of triplicate determinations;
  • FIG 8 represents the effect of DESC on the reversal of DFMO-induced growth inhibition by exogenous spermidine in ZR-75-1 cells.
  • Cells were incubated for 11 days in SD medium with the indicated concentrations of spermidine in the presence of 50 ⁇ M DESC (•), lmM DFMO (D), or the combination thereof ( ⁇ ), or in the absence of drugs
  • FIG 9 represents the chromatographic profile of DESC and its degradation products in IMEM or PBS.
  • DESC 50 ⁇ M was added to 1ml of IMEM containing 10% fetal bovine serum in the absence (A) or presence (B) of lmM aminoguanidine, or 1 ml PBS (C) in 24- well culture plates in the absence of cells. Media were analyzed after 20 minutes (solid lines) or 48 hours (dotted lines) of incubation at 37°C in 95% air: 5% CO 2 , water-saturated atmosphere for amine composition by ion-pair reversed-phase HPLC as described supra. Peaks 1 and 2 are degradation products of DESC, whereas peak 3 is a minor amount of DEASC initially present in the DESC preparation. Note the disappearance of peak 3 (DEASC) and the appearance of a shoulder (indicated by the arrow) at 42 minutes on the 48-hour profile in panel A; and
  • FIG 10 represents the time course of degradation of DESC in growth medium.
  • 50 ⁇ M DESC was added to 1ml of IMEM in 24-well culture plates and the content in DESC (O), compound 1 (Comp 1, •) and compound 2 (Comp 2, D) determined by
  • FIG 11. Structures of putrescine, of the natural polyamines spermidine and spermine, and of three cell-impermeant inhibitors of polyamine transport (DESC, DEASC and MESC).
  • FIG. 12 Structure and scheme for the synthesis of unmethylated spermine analogs as polyamine transport inhibitors with a linker attached via amide bonds to the polyamine chains (BS-3, BS-4, BS-5 and BS-6 compounds). The method of synthesis is described in greater detail in Example 1.
  • FIG 13 Initial route of synthesis of terminal C-methylated, dimeric spermine analogs as transport inhibitors with a linker attached via an alkyl bond to the polyamine chains (BMS-3, BMS-4, BMS-5 and BMS-6). The steps presented in this figure describe the complete route of synthesis leading to the precursor N 1 , N 4 , N 8 , N /2 -tetra (Boc)-l, 12- dimethylspermine-5-carbinol (XV).
  • FIG 16. The final step of the synthesis of BMS compounds (XX); the Boc- protected, cross-linked 1, 12-dimethylspermine dimer is deprotected to generate the BMS compounds.
  • BMS-3, BMS-4, BMS-5 and BMS-6 correspond to JV", N ⁇ -bis ([1, 12- dimethyl-spermine]-5-methyl)-diaminoalkanes where the diaminoalkane linker is 1,3- diaminopropane, 1 ,4-diaminobutane, 1,5-diaminopentane, and 1,6-diaminohexane, respectively.
  • FIG 17 A, 17B and 17C presents three classes of dimeric polyamine transport inhibitors according to the site of attachment of the linker (L) to the polyamine chain.
  • R H, methyl, ethyl, or propyl
  • R 2 H or methyl
  • L a chemical structure (the linker) connecting covalently the two polyamine chains via alkyl, amide, ether or thioether bonds with a substituent group (R 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain.
  • R 3 a chemical structure (the linker) connecting covalently the two polyamine chains via alkyl, amide, ether or thioether bonds with a substituent group (R 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain.
  • R, H, methyl, ethyl, or propyl
  • R 2 H or methyl
  • 2 ⁇ x ⁇ 5
  • L' a chemical structure (the linker) connecting covalently two polyamine chains via alkyl bonds with one of the two most internal amino groups of each polyamine chain.
  • R H, methyl, ethyl, or propyl
  • R 2 H or methyl
  • R3 a chemical structure (the linker) connecting covalently two polyamine chains via an alkyl, amide, ether or thioether bond with a substituent group (R3) attached on one carbon atom located between the two most internal amino groups of one polyamine chain, to one of the two most internal amino groups of the other polyamine chain via an alkyl bond.
  • R3 a chemical
  • R is H, methyl, ethyl or propyl;
  • R 2 is H or methyl;
  • R 3 is an alkyl, amide, keto, ether, thioether, phosphono or sulfonyl group;
  • x is greater than 2 and less than 5 (2 ⁇ x ⁇ 5), and the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y + ⁇ ⁇ 6).
  • the linker L is any chemical structure covalently linked to the R 3 groups and which prevents the uptake of the analog.
  • FIG 17B N-linked dimeric analogs.
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than 2 and less than 5 (2 ⁇ x ⁇ 5)
  • w is greater than 2 and less than 7 (2 ⁇ w ⁇ 7).
  • the linker L is any chemical structure covalently linked to one internal amino group of each polyamine chain and which prevents the uptake of the analog.
  • FIG 17C C-linked/N-linked mixed dimeric analogs.
  • R is H, methyl, ethyl or propyl;
  • R 2 is H or methyl;
  • x is greater than 2 and less than 5 (2 ⁇ x ⁇ 5), the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y + ⁇ ⁇ 6), and
  • w is greater than 2 and less than 7 (2 ⁇ w ⁇ 7).
  • the linker L is any chemical structure covalently linked to one internal amino group of one polyamine chain and to the R 3 of the other polyamine chain, and which prevents the uptake of the analog.
  • FIG 18. Initial route of synthesis of unmethylated, N 4 -alkylated dimeric spermine analogs (FIG 17B). Steps leading to the synthesis of the intermediate TV 1 -benzyl, N , N 12 - di(CBZ)-spermine.
  • FIG 19. Final steps for the synthesis of unmethylated, N 4 -alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXIX).
  • FIG 20 Initial route of synthesis of terminal C-methylated, ⁇ -alkylated dimeric spermine analogs (FIG 17B). Steps leading to the synthesis of the intermediate N a , N"-bis (N-[/V-Boc-3-amino, 3-methylpropyl], N-[4-aminobutyl])- ⁇ -diminoalkane.
  • alphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51 For the alphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • FIG 21 Final steps for the synthesis of terminal C-methylated, N'-alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXXVIII).
  • FOG 17B Final steps for the synthesis of terminal C-methylated, N'-alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXXVIII).
  • aliphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51 For the aliphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • FIG 22 Initial route of synthesis of 1,12-dimethylspermine dimers cross-linked through ⁇ -alkyl/S-alkyl attachments of the linker (FIG 17C). Steps leading to the synthesis of the intermediate N ⁇ f/7V-Boc-3-amino, 3-methylpropyl], N-[7V-FMOC-4- aminobutyl]), If-[5-(N N 4 , N 8 , N 12 -tetra (Boc)-spermine)-methyl]- ⁇ , ⁇ -diaminoalkane.
  • FIG 23 Intermediate route of synthesis of 1,12-dimethylspermine dimers cross- linked through N4-alkyl/5 -alkyl attachments of the linker (FIG 17C). Steps leading to the synthesis of the intermediate N a ([N -Boc-3-amino, 3-methylpropyl], N-[8-amino-5-aza- octanoyl]), W-[5-(N ⁇ , N 4 , N 8 , N 12 -tetra (Boc)-spermine)-methyl]- ⁇ , ⁇ -diaminoalkane.
  • N a [N -Boc-3-amino, 3-methylpropyl], N-[8-amino-5-aza- octanoyl]
  • FIG 24 Final route of synthesis of 1,12-dimothylspermine dimers cross linked through N4-alky 1/5 -alkyl attachments of the linker (FIG 17C represented by type compound XLV).
  • linker (CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • Sym-norspermidine, ornithine dihydrochloride and other reagents for organic syntheses were purchased from Aldrich (Milwaukee, WI) and Sigma (St. Louis, MO).
  • Reversed phase silica gel liquid chromatography was performed with a LichroprepTM RP- 18 C 18 silica gel column (40-63 ⁇ M ; BDH, St. Laurent, Qc, Canada) using a gradient of CH 3 CN:MeOH:H 2 O (25:35:40 to 50:30:20) as eluent. Homogeneity of synthetic products was assessed by thin-layer chromatography performed on 0.20 mm F 254 silica gel 60 plates or 0.25 mm F 254 S RP-18 reversed phase silica gel plates (E. Merck, Darnstadt, Germany).
  • FIR spectra were obtained on a Perkin-Elmer 1600 spectrophotometer (FTIR series) and were expressed in cm "1 .
  • FTIR series Perkin-Elmer 1600 spectrophotometer
  • 'H and 1 C NMR spectra were recorded with a Bruker AC/F 300 (300 MHz); 13 C were recorded at 75.47 MHz.
  • Chemical shifts ( ⁇ in ppm) were referenced to CDC1 3 (7.26 ppm for *H and 77.00 ppm for 13 C).
  • Mass spectra were recorded at the Mass Spectrometry Regional Center (University of Montreal, Montreal, Qc, Canada) by fast atomic bombardment mass spectrometry (FABMS) or liquid secondary ion mass spectrometry (LSMIS), using a VG AutoSpecQTM and a Kratos MS50 TCTA, respectively.
  • FABMS fast atomic bombardment mass spectrometry
  • LSMIS liquid secondary ion mass spectrometry
  • [2,3- 3 H(N]putrescine dihydrochloride (4.1 x 10 4 Cl/mol) and [l,8- 3 H(N)]spermidine trihydrochloride (1.5 x 10 4 Cl/mol) were obtained from Dupont-New England Nuclear (Lachine, Qc, Canada).
  • [5,8- 14 C]spermine tetrahydrochloride (108 Cl/mol)) was purchased from Amersham (Arlington Heights, IL).
  • DFMO was generously provided by the Marion Merrell Dow Research Institute (Cincinnati, OH).
  • Fetal bovine serum (FBS) and CosmicTM calf serum were from Hy clone (Logan, UT).
  • Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, iodoacetamide, 5,5'-dithio(2-nitrobenzoic acid) and 3,4-diaminobenzoic acid as well as tissue culture reagents were purchased from Sigma.
  • Ort/20-phthaldialdehyde was purchased from Fluka (Ronkonkoma, NY) and other reagents for high-performance liquid chromatography (HPLC) were from Fisher Scientific (Montreal, Qc, Canada) or
  • DESC was dissolved in 50 mM sodium phosphate bugger, pH 8.0, containing 250 mM dithiothreitol (DTT), and incubated for 30 minutes at 37° C in a water bath. The mixture was then loaded on a DowexTM 50W-X4 cation exchange column equilibrated with
  • ZR-75-1 human breast cancer cells and Chinese hamster ovary cells were obtained from the American Type Culture Collection (Rockville, MD).
  • ZR-75-1 cells were maintained in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, 1 mM sodium pyruvate, 15 mM Hepes, 10 nM 17 ⁇ - estradiol, and antibiotics [MEZR medium] (Huber, M. and Pouline, R. 1995. Cancer Res. ,
  • CHO-Kl cells were routinely grown in -Minimal Essential Medium supplemented with 10% CosmicTM calf serum in a 5% CO 2 humid atmosphere at 37°C.
  • ZR-75-1 cells were cultured in MEZR medium or in phenol red- free RPMI 1640 supplemented with 2mM L-glutamine, 1 mM sodium pyruvate, 15 mM
  • the effect of the transport inhibitors on cell growth was measured by incubating ZR-75-1 cells for 11 days in medium supplemented with antagonist, polyamines and/or 1 mM DFMO as indicated, followed by calorimetric determination of DNA content with 3,4-d]aminobenzaic acid (Simard, J., Dauvois, S., Haagensen, D.E., Levesque C, Merand, Y. and Labrie, F. 1990.
  • ZR-75-1 cells were plated in 100 mm culture dishes at 5 x 10 5 cells/dish in MEZR medium and grown for 5 days with medium changes every other day. Fresh MEZR medium containing the indicated concentration of transport antagonist was then added, plus or minus 200 ⁇ M cycloheximide (CHX), and cells were incubated for 1 or 6 hours. Medium was then removed, cell monolayers rinsed twice with 10 ml of ice-cold
  • Tris-DTT buffer 50 mM Tris/HCl, 0.1 mM EDTA, 5 mM DTT, pH 7.5
  • samples were first quickly thawed and incubated for 15 minutes at 37 °C. Trichloroacetic acid was then added to
  • DTT-containing samples to a final concentration of 10% (wt v). Samples were dispersed for 2 minutes in a sonicating water bath, and pelleted in a microcentrifuge for 5 minutes.
  • the trichloroacetic acid-insoluble pellet was solubilized in 300-500 ⁇ l of 1 N NaOH and used to determine protein content using bovine serum albumin (fraction V) as standard.
  • Polyamine contents were then analyzed by ion pair reverse-phase HPLC with flurometric detection after postcolumn derivatization with o-phthaldialdehyde as described (Pegg, A.E.,
  • DESC stability was tested by incubating the compound dissolved (at 50 ⁇ M) in PBS or in IMEM medium containing 10% (v/v) fetal bovine serum plus or minus 1 mM aminoguanidine in a humid 5% co2 atmosphere at 37°C and in the absence of cells. At indicated times, trichloroacetic acid was added to aliquots of this solution to a final concentration 10% (w/v) and the samples directly analyzed by HPLC as above.
  • the rate of putrescine and spermidine transport was determined in ZR-75-1 cells incubated in serum-free RPMI 1640 medium as described (Lessard, M., Zhao, C, Singh, S.M. and Poulin, R. 1995. J. Biol. Chem. 270: 1685-1694), using [ 3 H jputrescine (30 Ci/mol) and [ 3 H] spermidine (20 Ci/mol), respectively as substrates for a 20 minute-assay period.
  • Spermine uptake was similarly determined, using 1 ⁇ M [ 14 C]spermine (32 Ci/mol) as substrate.
  • Uptake activity was expressed per amount of DNA as flurometrically determined using 3,4-diaminobenzoic acid (Simard, J., Dauvois, S., Haagensen, D.E., Levesque, C, Merand, Y. and Labrie, F. 1990. Endocrinology, 126: 3223-3231).
  • spermidine uptake activity in CHO-Kl cells 80% confluent cell monolayers were rinsed twice with PBS and incubated for 20 minutes at 37 °C in 400 ⁇ l of buffer A (20 mM Tris-HCl, pH 7.4; 0.42 mM CaCl 2 ; 0.41 mM MgSO 4 ; 103 mM NaCL; 5.7 mM KC1; 1.1 mM D-glucose) containing 5 ⁇ M [ 3 H] spermidine (20 Ci/mol). Cell cultures were then washed twice with 1 ml PBS containing 5.7 mM .yym-norspermidine.
  • K ?? K, and V max values were then estimated by Lineweaver-Burke analysis.
  • Kj values were also estimated by measuring uptake activity in the presence of logarithmically increasing concentrations of antagonist, and using the Cheng-Prusoff equation (Cheng, Y.-C. and Prusoff, W.H. 1973. Biochem. Pharmacol. 22: 3099-3108) by iterative curve fitting for a sigmoidal curve.
  • Cheng-Prusoff equation Cheng-Prusoff equation
  • the time course of intracellular accumulation of spermidine in the presence of transport antagonists was determined by incubating ZR-75-1 cells in 24-well plates with DESC (50 or 200 ⁇ M) or MESC (200 ⁇ M) in dissolved in MEZR medium containing 5 ⁇ M [ 3 H] spermidine in the presence or absence of cycloheximide (CHX, 200 ⁇ M), and harvesting at the indicated times for the determination of intracellular radioactive contents, as described above for polyamine uptake assays.
  • DEASC reversed-phase liquid chromatography on C w silica gel.
  • Table I summarizes the K, values determined for DESC, MESC and DEASC toward putrescine, spermidine and/or spermine uptake, in relation with the mutual transport interactions between the latter substrates.
  • K values of the three spermine conjugates with respect to putrescine uptake were 3-fold to 5-fold higher than for spermine uptake, unlike spermidine and spermine which both inhibited the uptake of either substrate with similar potency, and with a K, roughly equal to their K,,, as substrate.
  • MESC was a less potent inhibitor of diamine and polyamine transport than DESC and DEASC suggested that the nature of the side chain strongly influences the interaction of these compounds with the carrier.
  • the thiol side chain of MESC was thus derivatized with substituting groups of different sizes and charges through thioether linkage with three different iodoacetamides, namely LY iodoacetamide, ASIB and iodoacetamide itself, and the ability of the resulting complexes (MESC-LY, MESC- ASIB, and MESC-acetamide, respectively) to inhibit spermidine uptake was then evaluated.
  • ZR-75-1 cells The ability of ZR-75-1 cells to accumulate DESC and MESC was determined. Since DESC was eluted as a late, broad peak in the HPLC system used, DTT was added to cell extracts to reduce DESC to MESC and decrease the detection threshold. Results are shown in Table II. ZR-75-1 cells were incubated for 1 or 8 hours in MEZR medium in the presence of 50 or 200 ⁇ M DESC or MESC prior to determination of polyamine contents. CHX was added at 200 ⁇ M where indicated. Other details are provided under "Materials and Methods.” Values are the mean ⁇ SD of triplicate determinations from 2 independent experiments.
  • spermidine accumulation in the presence of either inhibitor followed a pattern similar to that of control cells, i.e. a rapid phase during the first 60 minutes, followed by a much slower rate of accumulation thereafter, which was nearly independent of antagonist concentration.
  • This pattern suggests that even cellular levels of newly internalized spermidine as low as 20% of those found under control conditions, e.g., in cells treated with 200 ⁇ M DESC, may induce a near maximal degree of feedback repression of polyamine transport. Nevertheless, even a 40-fold excess of the most potent antagonist (i.e. 200 ⁇ M DESC) only descreased net spermidine accumulation by only 50% after 6 hours.
  • the most potent antagonist i.e. 200 ⁇ M DESC
  • DESC was only mildly growth inhibitory at 50 ⁇ M, there was an abrupt, aminoguanidine-resistant increase in toxicity at 200 ⁇ M.
  • spermine was acutely cytotoxic at 50 ⁇ M, an effect that was only partly prevented by aminoguanidine.
  • MESC was considerably less toxic than its dimer, with a 35% decrease in cell growth at 200 ⁇ M which was not blocked by aminoguanidine.
  • DESC and to a much lesser degree, its thiol monomer MESC, are cytotoxic toward breast cancer cells at high concentrations through a mechanism that does not involve BSAO.
  • DESC is indeed a potent antagonist of polyamine accumulation
  • the slow residual uptake that occurred even at a 40-fold molar excess of inhibitor might be sufficient to counteract polyamine depletion by inhibitors of polyamine biosynthesis.
  • This possibility was assessed by comparing the ability of DESC to prevent the reversal of DFMO-induced growth inhibition by increasing concentrations of exogenous spermidine. At concentrations superior to 0.3 ⁇ M, spermidine inhibited ZR-75-1 cell proliferation by up to 20% (Fig. 8).
  • DESC solutions (20 ⁇ M) made in PBS or in sterile IMEM medium enriched with 10% (v/v) FBS were incubated for 20 minutes or 48 hours under cell-free conditions at 37° C in a humid 5% CO 2 atmosphere, and the polyamine analog was then analyzed by ion-pair reversed-phase HPLC. After 48 hours, degradation of DESC to two new amine-containing derivatives occurred in IMEM (Fig. 9 A, B) but not in PBS (Fig.
  • DESC a novel type of spermine derivative
  • DESC is shown to be endowed with high affinity for the polyamine transport system while being highly resistant to cellular uptake.
  • the combination of these two attributes confers unique characteristics to DESC as a pure competitive antagonist of polyamine uptake.
  • At least one mammalian glucose transporter exists as a tetrameric complex in its native form (Hebert, D.N. and Carruthers, A. 1992. J Biol. Chem.267: 23829-23838; Gould, G.W. and Holman,
  • dimerization of MESC into DESC could impose conformational constraints (e.g. due to electrostatic repulsion) that would favor recognition of the polyamine binding site of the carrier by each of the symmetrical spermine moieties.
  • MESC thioethers as diverse in size as MESC-LY, MESC-ASIB, or MESC- acetamide had Kj values virtually identical to that of MESC, indicating that the thiol group of MESC does not specifically determine its lower affinity as a polyamine transport inhibitor as compared with DESC.
  • MESC- cysteamine mixed disulfide DEASC
  • MESC and DESC behaved like pure competitive inhibitors of putrescine transport. Since the interaction of DESC or MESC with the polyamine transporter was strictly competitive, and because DEASC exhibits higher affinity than MESC as an inhibitor of diamine and polyamine transport, the spermine head and the cysteamine side chain of DEASC might be respectively responsible for the competitive and non-competitive components of its transport inhibition.
  • DESC The biochemical properties of DESC clearly illustrate that the binding affinity of a compound can be dissociated from its ability to serve as a substrate for the polyamine transporter.
  • the large size of DESC cannot be the main factor preventing its internalization through the channel-like portion of the transporter since MESC was also virtually impermeant.
  • MESC was also virtually impermeant.
  • the mere attachment of an amido side chain on the spermine backbone would appear to be responsible per se for the impaired internalization of MESC and its derivatives.
  • N 4 -alkylated spermidine derivatives are far better competitors of spermidine uptake than their iV-acyl counterparts in mouse leukemia cells, in support of the notion that charged secondary amino groups are important in the interaction with the polyamine carrier (Porter, C.W., Cavanaugh, P.F., Jr., Stolowich, N., Ganis, B., Kelly, E., and Bergeron, R.J. 1985. Cancer Res. 45: 2050-2057).
  • the latter argument cannot account for the fact that long-chain aliphatic , ⁇ -diamines with at least 6 to 7 methylene groups have an affinity comparable to that of spermidine (Lessard, M., Zhao, C, Singh, S.M. and Poulin, R., 1995. J. Biol. Chem. 270: 1685-1694, Bergeron, R.J. and
  • chlorambucil-spermidine which bears a N-propyl chlorambucil carboxamide side chain on the central nitrogen of spermidine, is a good substrate of the polyamine transport system, with a K m averaging that of spermidine
  • MESC-ASIB might serve as a photoaffmity label to detect polyamine-binding proteins, including the polyamine carrier. Experiments are currently conducted with 125 I-labeled MESC-ASIB to assess its usefulness as a probe to identify the mammalian polyamine transporter.
  • the basic features of this molecule should be useful for the design of potent transport inhibitors with minor non-specific effects on cell viability.
  • the inherent structural features of DESC that confer its high affinity and resistance to uptake should thus provide a useful framework for the design of potent irreversible inhibitors of polyamine transport, which could incorporate an alkylating group such as that used in the design of specific suicide substrates of mammalian glucose transporters (Clark, A.E., and Holman, G.D. 1990. Biochem. J. 269: 615-622; Lehmann, J., and Scheuring, M. 1995. Carbohydrate Res. 276: 57-74)].
  • Polyamine derivatives (natural or synthetic) comprising sulfur in the side chain have been made, because they conducted to the formation of dimers simply by forming a disulfide bridge. By-products which are not dimers have also shown an activity. However, it will be readily apparent to those skilled in the art that compounds being more stable than those containing sulfur atoms are contemplated. Therefore, the side chains used for increasing the affinity of the derivatives for a polyamine transporter and/or as substrates for labeling molecules and/or as a spacer in the making of a dimer can be varied to optimize the characteristics of the derivatives of the present invention.
  • Eflornithine Eflornithine. These molecules are based on the overall design of a prototype, 2, 2'- dithiobis(N-ethyl-spermine-5-carboxamide) (DESC). DESC has recently been reported to act as a competitive and potent antagonist of polyamine uptake in leukemia and breast cancer cells. DESC is proposed here to potentiate the chemotherapeutic efficacy of DFMO. While not intending to be limited to any particular theory, it is proposed that such effect is provided by preventing the replenishment of DFMO-treated tumor cells with polyamines from exogenous sources. Structural modifications to the molecule will improve it to a pharmacologically useful compound.
  • DESC 2, 2'- dithiobis(N-ethyl-spermine-5-carboxamide)
  • DESC analogs Two types will be synthesized, and characterized for their ability to inhibit polyamine transport and to enhance the therapeutic action of DFMO in various tumor cell types, including animal models.
  • the first type of analogs will be simply obtained by substituting the original cystamine side chain of DESC with ⁇ , ⁇ -diamine cross-linkers of varying length. The synthesis of these analogs will help in the short term to optimize the length of the cross-linker chain, and to rapidly evaluate their relative ability to potentiate DFMO action in vitro.
  • the second type of analogs will be made according to a new route of synthesis to introduce methyl groups at the extremities of the spermine- like backbone, and will also incorporate alkylation instead of acylation of the aliphatic, ⁇ , ⁇ -diamine cross-linker in order to improve their affinity for the polyamine transport system, their potency as antagonists of uptake and as enhancers of DFMO therapeutic action.
  • the pharmacological evaluation of the second-type analogs will be conducted in a standard mouse model bearing L1210 leukemia tumor cells treated with DFMO.
  • MESC is significantly internalized by human breast cancer cells or mouse leukemia cells at concentrations that saturate the polyamine carrier, indicating that they are essentially membrane-impermeant (21).
  • the combination of high affinity and lack of carrier-mediated permeation of DESC provided the basis for a novel design of pure polyamine transport antagonists that could be used in combination with DFMO to enhance polyamine depletion in tumor cells exposed to physiological levels of exogenous polyamines.
  • DESC was designed for biochemical use. It was found to degraded in physiological media due to thiol-disulfide reaction with compounds such as L-cystine. DESC cannot efficiently counteract the ability of exogenous spermidine to reverse DFMO-induced cytostasis in breast cancer cells as a result ofthis instability (21). DESC is also subject to attack by serum amine oxidase (SAO), an ubiquitous plasma enzyme which oxidatively deaminates aminopropyl groups, albeit to a much lesser degree than the parent compound, spermine. Modifications that further improve the design of DESC analogs that are part of particular embodiments of the present invention are:
  • DESC analogs are prepared with unmodified spermine backbones but different side chain lengths as lead compounds to guide us in the design of methylated analogs described herein. This series of compounds will be synthesized in order to: (i) Perform a structure-function study in the short-term to determine the optimal length of the cross-linker for inhibition of polyamine uptake.
  • BS-3, BS-4, BS-5 and BS-6; Fig. 12 The kinetic properties of these DESC analogs (abbreviated as BS-3, BS-4, BS-5 and BS-6; Fig. 12), as inhibitors of polyamine transport will be determined by uptake assays of radiolabeled putrescine, spermidine and spermine, according to procedures in Huber et al. (1996), J. Biol. Chem., 271: 27556-27563, which is specifically incorporated herein by reference. These structures are shown below.
  • n 3, 4, 5 or 6.
  • These compounds are expected to be stable under cell culture conditions in the presence of aminoguanidine, a SAO inhibitor (13, 28, 40, 46, 49, 66, 67).
  • SAO inhibitor 13, 28, 40, 46, 49, 66, 67.
  • These polyamine transport inhibitors will be evaluated using ZR-75-1 human breast cancer cells and L1210 mouse leukemia cells. Briefly, the rate of cell proliferation will be determined in ZR-75-1 and L1210 cells grown in the presence or absence of DFMO (1 and 5 mM, respectively), and of the transport inhibitor candidate to be analyzed, in the presence of increasing concentrations of putrescine or spermidine.
  • the ability of the transport antagonist to prevent the reversal of DFMO-induced growth inhibition by exogenous putrescine or spermidine will provide a valid measurement of the pharmacological potential of these compounds as enhancers of DFMO action in vivo.
  • These studies will also include (a) dose-response experiments to evaluate the cytotoxicity of these analogs and the optimal concentration for their use as inhibitors of polyamine uptake, and (b) measurement of the uptake of the transport inhibitors during incubation with tumor cells by HPLC, along with their effect on polyamine pools.
  • This improved scheme also includes the use of mono- FMOC-protected diamines as building blocks for cross-linking the dimethylspermine-5- methyl precursors, as described above for the unmethylated DESC analogs.
  • the resulting compounds are abbreviated as BMS-3, BMS-4, BMS-5 and BMS-6 (Fig. 16; compounds XXa to XXd).
  • n 3, 4, 5 or 6.
  • Protocol 1 - Toxicity will first be determined by single i.v. and i.p.injections of logarithmically increasing drug concentrations to mice and estimating the LD 50 . Blood samples will be taken at intervals to measure the plasma drug concentration by ion pairing reverse-phase HPLC (22, 23). Body weight and liquid consumption will also be monitored for 10 days, at the end of which period animals will be sacrificed to evaluate the incidence of liver and kidney damage. A similar experiment will be conducted by dissolving the drug in the drinking water with free access to the animals.
  • mice will be injected with L1210 cells, with concomitant treatment with DFMO or vehicle, plus or minus 2 different sublethal doses of the transport antagonist on a daily schedule. Oral, i.v. and i.p. routes will be compared for the transport antagonist. Survival will be evaluated for up to 120 days, with regular body weight measurements and blood sampling to determine the steady-state plasma concentrations of inhibitor. L1210 cells are strongly immunogenic tumors and cured animals develop extended immunity against this leukemia (1). Thus, to evaluate the curative potential of the drug combination, survivors will be rechallenged with L1210 cells in the absence of treatment and survival monitored. EXAMPLE 9
  • the present example demonstrates the utility of the present invention with the use of compounds that are analogs of spermine that include two chains connected to one another through a linker.
  • the linker molecule that attaches the two spermine chains may be any spacer chain that is capable of bridging the polyamine chains.
  • the two chains may attach to the linker at an internal C atom or an N group within the chain. It is also possible for one chain to be connected to the linker through one of its carbon molecules, while the second chain attaches to the linker molecule through an N group within its chain.
  • the general structure of compounds claimed include the following characteristics:
  • the central carbon chain of the spermine backbone can have between 3 and 7 methylene groups or carbon atoms. This is the range of central chain length that can be accommodated with good affinity by the mammalian polyamine transporter (81).
  • Each methylene group of the polyamine chains can be modified by methyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter.
  • the linkage between the polyamine chains and the spacer may comprise any type of linkage compatible with a Kj ⁇ 20 ⁇ M (relative to spermine) for the resulting inhibitor, such as direct alkyl substitution or ether group on the central methylene groups (Structure 1), or alkylation on the secondary amino (Structure 2) groups of the polyamine chain.
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • the linker connecting covalently the two polyamine chains via alkyl, amide, ether or thioether bonds with a substituent group (R 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain.
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • w is greater than 2 and less than 8 (2 ⁇ x ⁇ 8)
  • the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • w is greater than 2 and less than 8 (2 ⁇ x ⁇ 8)
  • the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • Linker (L) can be of any nature or chain length, as long as the total mass of the final structure does not exceed 3,000. These molecules may in other embodiments be described as having a total mass of between about 50 to about 2,5000, or about between 500 to about 1500 or about 1,000 as a total mass.
  • linkers may comprise an alkyl, an ether, a thio ether, an amide, phosphono, keto, amine, and sulfonyl or a combination thereof.
  • the alkyl linker may comprise a carbon chain by a length of 2 to 50 carbons.
  • the carbon chain will have a length of between 5 to about 25 carbons, or between 10 and 20 carbons, or in even other embodiments, the carbon length of 2 to about 15 or 12 carbons.
  • the FMOC group of the latter compound is removed with 20% piperdine/DMF, and the resulting N 1 , N 4 , N8, N /2 -tetra-Boc-spermine- 5(N- ⁇ -aminoalkyl) carboxamide (VI, FIG 12) is then reacted with the acid chloride form of N', N 4 , N8, N ;2 -tetra-Boc-5-carboxyspermine (IV, FIG 12).
  • the latter compound is then deprotected with HCl/CH 3 COOH to obtain the corresponding N", N ⁇ -bis(spermine-5-oyl)- diaminoalkane, the desired transport inhibitor (VII, FIG 12).
  • BS-3, BS-4, BS-5 and BS-6 correspond to the forms where the diaminoalkane linker is 1,3-diaminopropane, 1, 4-diaminobutane, 1, 5- diaminopentane and 1 , 6-diaminohexane, respectively.
  • the spacer is going to be alkylated to the polyamine chain, the carboxyl group used as an acceptor in an amidation reaction is first reduced to an alcohol with LiAlH 4 . After protecting the amine groups with carbobenzoxy groups, the alcohol is then converted to a bromide with Pbr 3 .
  • the resulting CBX-protected spermine bromide is then reacted with a diamine spacer with a 2:1 stoichiometry to generate the CBZ-protected spermine dimer.
  • This dimer is finally deprotected by catalytic hydrogenation with Pd/C (82) to generate the unmethylated spermine dimer (the transport inhibitor).
  • Pd/C the transport inhibitor
  • the alcohol obtained as above is then converted to an alkoxide with sodium metal, and then reacted with an alkyl dihalide (e.g.
  • Methylated spermine analogs (FIG 17A): For example, ornithine methylester (X, FIG 13) is synthesized as described (89) and is diamidated with two equivalents of 3- azidobutyric acid (XII, FIG 13) using DCC/OHB t (90) to generate N 1 , N'-bis (3- azidobutyryl)-ornithine methylester (XIII, FIG 13). The latter is then reduced using
  • N-benzyl-l,3-diaminopropane is then N-alkylated with 3- bromobutyronitrile to generate N 1 -benzyl, N 3 -(3-cyanopropyl)-l ,3-diaminopropane
  • N 1 -benzyl, N 3 -(3-cyanopropyl)-l,3,-diaminopropane is protected with a Boc group (86) to generate N 1 -benzyl, N 3 -Boc, N 3 -(3-cyanopropyl)-l,3,- diaminopropane (XXIII, FIG 18)
  • N 1 -benzyl, TV 3 -Boc, ⁇ 3 -(3-cyanopropyl)-l,3,-diaminopropane is reduced to N 1 -benzyl, TV-Boc-spermidine ( XXIV, FIG 18) by catalytic hydrogenation with Raney nickel (84).
  • N 1 -benzyl, N -Boc-spermidine is then cyanoethylated with acrylonitrile to generate N 1 -benzyl, N'-Boc, ⁇ -cyanoethyl- -spermidine, and reduced to N 1 -benzyl, N'-Boc-spermine by catalytic hydrogenation with Raney nickel (84) (XXV, FIG 18).
  • N 1 -benzyl, N -Boc-spermine are protected with CBZ groups as described (87) to generate N 1 -benzyl, N'-Boc, N 8 , N 12 -di(CBZ)-spermine (XXVI, FIG 18).
  • N 1 -benzyl, N 4 -Boc, N 8 ,N 12 -di(CBZ)-spermine is then deprotected to
  • N 1 -benzyl, N 8 , N I2 -di(CBZ)-spermine can then be cross-linked with an ⁇ , ⁇ -dibromoalkane of the desired chain length to generate the corresponding bis(N'-benzyl, N 8 , N 12 -di(CBZ)-spermine) dimer (XXVIII, FIG 19), which is then deprotected by catalytic hydrogenation with Pd/C (87) to generate the unmethylated, N-alkylated spermine dimer (the transport inhibitor) (XXIX, FIG 19).
  • XXIX the transport inhibitor
  • N 1 -(N-Boc-3 -aminobutyryl), N*-FMOC- 1 , 4-diaminobutane is then reduced to N 1 -Boc-N 8 -FMOC-l-methylspermidine with BH 3 /THF (88) (XXXII, FIG 20).
  • XXXII XXII
  • Dimeric polyamine transport inhibitors of a different type can be generated by cross-linking one polyamine chain to a linker through a N-alkyl bond as in Examples 3 and
  • N'-Boc-N ⁇ FMOC-l-methylspermidine (XXXII, FIG 20), obtained as described above (Example 4, steps a to b), is N'-alkylated using an ⁇ - bromoalkylphthalimide of the desired length as described (92), to generate the corresponding N'-Boc, N -alkylphthalimide, N -FM ⁇ C-1 -methylspermidine (XXXIX, FIG 22).
  • Gastrointestinal Tract edited by R. H. Dowling, U. R. F ⁇ lsch and C. Loser. Dordrecht: Kluwer Academic Publ., 1992, p. 435-445.

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Abstract

La présente invention concerne la conception, la synthèse et l'usage thérapeutique d'une variété de nouveaux inhibiteurs du transport de la polyamine. La caractéristique majeure de cette catégorie d'inhibiteurs du transport est qu'ils contiennent un lieur ou chaîne latérale qui empêche l'assimilation de polyamines et contrigue à conjuguer des analogues de polyamine pour former des dimères ayant un pouvoir inhibiteur élevé contre l'assimilation de la polyamine. Ces nouveaux composés présentent des caractéristiques qui ont pour fonction, d'une part, d'optimiser leur stabilité chimique et métabolique et leur capacité de liaison au transporteur de la polyamine et, d'autre part, de réduire au minimum leur toxicité en empêchant leur absorption par les cellules. L'objet desdits inhibiteurs est d'empêcher l'assimilation ou la récupération de polyamines en circulation par des cellules proliférant rapidement, telles que les cellules tumorales, afin de renforcer l'effet d'inhibiteurs thérapeutiques de la biosynthèse de la polyamine, tels que l'eflornithine.
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WO1999054283A1 (fr) * 1998-04-21 1999-10-28 Universite Laval Inhibiteurs du transport des polyamines
WO2000034226A1 (fr) * 1998-12-10 2000-06-15 Universite Laval Inhibiteurs de transport de polyamines
US6949679B1 (en) 1998-04-21 2005-09-27 Universite Laval Polyamine transport inhibitors
US7425579B2 (en) 1998-04-21 2008-09-16 Universite Laval Methods for inhibiting activity of polyamine transporters
EP3473247A1 (fr) 2009-07-16 2019-04-24 Pathologica LLC Composition pour administration par voie orale comprenant la mgbg pour utilisation dans le traitement de la sclérose en plaque
EP3831372A1 (fr) 2013-01-08 2021-06-09 Pathologica LLC Mitoguazone pour prévenir la rechute ou la progression de la sclérose en plaques

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WO2003015763A1 (fr) 2001-08-13 2003-02-27 Board Of Regents, The University Of Texas System Chimiotherapie adjuvante pour gliomes anaplasiques
US20030129208A1 (en) * 2002-01-07 2003-07-10 Alberts David S. Topical application of alpha-DFMO and anti-inflammatory drug for treatment of actinic keratoses
WO2020236562A1 (fr) 2019-05-17 2020-11-26 Cancer Prevention Pharmaceuticals, Inc. Méthodes de traitement de la polypose adénomateuse familiale

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999054283A1 (fr) * 1998-04-21 1999-10-28 Universite Laval Inhibiteurs du transport des polyamines
US6949679B1 (en) 1998-04-21 2005-09-27 Universite Laval Polyamine transport inhibitors
US7425579B2 (en) 1998-04-21 2008-09-16 Universite Laval Methods for inhibiting activity of polyamine transporters
WO2000034226A1 (fr) * 1998-12-10 2000-06-15 Universite Laval Inhibiteurs de transport de polyamines
EP3473247A1 (fr) 2009-07-16 2019-04-24 Pathologica LLC Composition pour administration par voie orale comprenant la mgbg pour utilisation dans le traitement de la sclérose en plaque
EP3831372A1 (fr) 2013-01-08 2021-06-09 Pathologica LLC Mitoguazone pour prévenir la rechute ou la progression de la sclérose en plaques

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